Hemorrhage is a major cause of the morbidity and mortality associated with traumatic cardiovascular injury, whether this injury is sustained on the battlefield, during a vehicular collision, or in the operating room. Rapid control of hemorrhage, especially in a chaotic setting, would undoubtedly reduce this morbidity and mortality. One very promising method for such control involves mimicry of the body's own hemostatic system: a concentrated solution of fibrinogen (the precursor of the protein matrix of blood clots) and thrombin (the mammalian enzyme that catalyzes fibrin formation) is applied topically to a bleeding wound. The separate but simultaneous application of substrate (i.e., fibrinogen) and catalyst (i.e., thrombin) results in the rapid formation of a fibrin clot at the site of application. The fibrin thus formed promotes not only local hemostasis but also wound healing. While this approach has been used with some success, it does not effectively control bleeding from larger wounds. In the body's natural clotting system, platelets work synergistically with fibrin to control hemorrhage. The physical size of the platelets helps plug the wound opening. The use of fibrin without platelet bodies is not nearly as effective in controlling bleeding as are the two together, particularly from larger wound sites.
The use of platelets to control bleeding, however, presents some very practical problems. For example, platelets have a very short shelf life and require rigorous storage temperature and mixing conditions to maintain their efficacy in vivo. This makes platelets impractical for use in large scale emergency trauma situations, particularly in non-hospital settings. Therefore, it would be very useful to develop a composition which mimics the blood clotting efficacy of platelets, but which does not have the same storage problems which they typically present.
When used together with thrombin as catalyst, fibrinogen effectively limits bleeding from smaller wounds. See, for example, Gibble and Ness, Transfusion 30 (1990) 741-747; Kram, Shoemaker, Clark, Macabee and Yamaguchi, Am. Surg. 57 (1991) 381-384; and Redl, Schlag and Dinges, Thorac. Cardiovasc. Surgeon 30 (1982) 223-227. See also, U.S. Pat. No. 4,647,536, Mosbach, et al, issued Mar. 3, 1987, which describes the encapsulation of viable animal or plant cells in polymer beads made from agar, agarose or fibrinogen; and European Patent Application 0 485 210, published May 13, 1992, which describes fibrous protein membranes, at least a portion of which is formed from fibrin, used to dress wounds, inhibit bleeding and deliver drugs to the wound site.
In addition, liposomes are very well known for the delivery of pharmaceutical actives to various sites in the human body. For example, U.S. Pat. No. 4,394,448, Szoka, Jr., et al, issued Jul. 19, 1983, describes lipid vesicles which encapsulate DNA or DNA fragments and are used to insert those DNA materials into living cells.
U.S. Pat. No. 5,264,221, Tagawa, et al, issued Nov. 23, 1993, describes drug-containing liposomes which have specifically defined targeting agents on their surface. These targeting agents are thiol-containing proteins and residues of thiol-containing compounds which include a polyalkylene glycol moiety bound to a maleimide residue on the surface of the liposome.
U.S. Pat. No. 5,283,122, Huang, et al, issued Feb. 1, 1994, describes fused liposomes and their use as delivery vehicles for pharmaceutical agents. An acid-induced liposome fusion procedure is disclosed.
PCT Patent Application WO 92/1447, Baxter International, published Sep. 3, 1992, describes liposomes used for drug delivery. The surface of those liposomes contains specific residues (i.e., glutaraldehyde and water-soluble carbodiimide) which are crosslinked to targeting agents such as gelatin, collagen, and hyaluronic acid. See also, PCT Patent Application WO 92/14445, Baxter International, published Sep. 3, 1992.
The use of platelet-derived proteins in liposomes has also been disclosed. Baldassare, et al, J. Clin. Invest. 75 (1985), 35-39, describes the incorporation of platelet glycoproteins IIb and IIIa into phospholipid vesicles. It was found that those proteins in the vesicles have similar physiological properties as they do in platelets. Rybak, et al, Biomat., Art. Cells & Immob. Biotech., 21(2), 101-118 (1993), describes the incorporation of an undefined mixture of platelet proteins into liposomes. This product was shown to have an effect in stopping wound bleeding. Neither of these papers discuss the incorporation of fibrinogen into any liposome structures.
Although not previously suggested by the art, the incorporation of fibrinogen into a liposome structure could provide many advantages. For example, assuming the liposomes could be coated with functional fibrinogen, they could provide rapid, biomimetic control of wound hemorrhage even in the chaotic setting of a trauma scene. These liposomes could not only provide hemostatic, but also wound healing, benefits. Large quantities of the liposomes could be produced at low cost and on demand. They could be more effective at facilitating hemostasis than the fibrin glues described in the art, without having the storage stability and logistical problems inherent in the use of platelets as a hemostatic agent. They could be stored indefinitely at room temperature in a lyophilized state. Once reconstituted in aqueous phase, their shelf life could be made indefinite by addition of antibiotics to the storage medium. In addition, use of such liposomes could be virtually without risk of blood-borne infection to the recipient.
Unfortunately, conventional approaches for binding proteins to liposomes do not work effectively with fibrinogen to provide a final product which has adhesive properties. Most of these approaches involve the use of a bifunctional reagent that cross-links a reactive group contributed by a liposomal component to a reactive group contributed by the protein. In the case of fibrinogen, such approaches do not yield liposomes coated densely with demonstrably functional protein. Acylation of the fibrinogen prior to incorporating it into liposomes is also problematic. In the absence of liposomes or other amphiphilic/hydrophobic surfaces, the acylation of fibrinogen yields an insoluble, amorphous material unsuitable for coating liposomes.
The present invention relates to a method for effectively incorporating fibrinogen onto the surface of liposome-like (e.g., cell membrane) structures or any amphiphilic or hydrophobic surface. It further encompasses the fibrinogen-coated liposome structures, themselves, and pharmaceutical compositions containing those structures. Those compositions can be used as platelet-like biochemical hemostats, drug delivery systems which target sites of inflammation and/or clotting, reagents for the imaging of clot-containing lesions, reagents for clinical, clot-based coagulation assays, a bioadhesive reagent to introduce molecules into cells or facilitate adhesion between chemical reactants, an adhesive reagent for facilitating chemical reactions between environmentally-incompatible reactants, a vaccine component or an adjuvant for vaccines, a reagent for modifying the lipid composition of biological membranes, and a reagent for transfection of genes into target cells.